Cephalosporin compounds

ABSTRACT

A cephalosporin compound of the formula: ##STR1## wherein R 1  is hydrogen or lower alkyl; 
     R 2  is acetoxy or (1-methyl-1H-tetrazol-5-yl)thio, and 
     R 3  is carboxy; or 
     R 2  is a group of the formula: ##STR2##  R 3  is --COO -  and Y is hydrogen, hydroxymethyl or carbamoyl; and 
     n is an integer of 2 or 3, 
     or a pharmaceutically acceptable salt thereof and processes for their preparation are disclosed. Said cephalosporin compound is useful as an antimicrobial agent.

This application is a divisional of application Ser. No. 835,886 filedon Mar. 4, 1986 which is a divisional of application Ser. No. 516,053filed on July 21, 1983 (now U.S. Pat. No. 4,598,075).

This invention relates to a novel cephalosporin compound and processesfor preparing the same. More particularly, it relates to a compound ofthe formula: ##STR3## wherein R¹ is hydrogen or lower alkyl;

R² is acetoxy or (1-methyl-1H-tetrazol-5-yl)thio, and R³ is carboxy; or

R² is a group of the formula: ##STR4## R³ is --COO⁻ and Y is hydrogen,hydroxymethyl or carbamoyl; and n is an integer of 2 or 3,

or a pharmaceutically acceptable salt thereof. A pharmaceuticalcomposition for use as an antimicrobial agent which comprises thecephalosporin compound (I) or a salt thereof as an active ingredienttogether with an inert carrier therefor is also provided in the presentinvention.

The new cephalosporin compound (I) of the present invention and apharmaceutical salt thereof show potent antimicrobial activity against awide variety of microorganisms including gram-positive and gram-negativebacteria and are useful as anti-bacterial agents, as nutritionalsupplements in animal food or as chemotherapeutic agents in warm-bloodedanimals, including man, in the treatment of infectious diseases causedby said gram-positive and gram-negative bacteria.

Among the compounds of the present invention, a preferred subgenusincludes those of the formula (I) in which R¹ is hydrogen, lower alkylsuch as methyl, ethyl or propyl; R² is acetoxy or(1-methyl-1H-tetrazol-5-yl)thio, and R³ is carboxy; or R² is a group ofthe formula: ##STR5## R³ is --COO⁻ and Y is hydrogen, hydroxymethyl orcarbamoyl; and n is an integer of 2 or 3. A more preferred subgenusincludes those of the formula (I) in which R¹ is hydrogen or methyl, R²is (1-methyl-1H-tetrazol-5-yl)thio, and R³ is carboxy; or R² is a groupof the formula: ##STR6## R³ is --COO⁻ and Y is hydrogen,4-hydroxymethyl, 3-hydroxymethyl or 4-carbamoyl; and n is an integer of2 or 3. A further preferred subgenus are those of the formula (I) inwhich R¹ is hydrogen, R² is a group of the formula: ##STR7## R³ is--COO⁻, Y is hydrogen and n is the integer of 2. The most preferredcompound is the levorotatory isomer of the compound (I) in which R¹ ishydrogen, R² is a group of the formula: ##STR8## R³ is --COO⁻, Y ishydrogen and n is the integer of 2. In addition, the structural formula(I) shown above intended to show that the isomeric configuration of theoxyimino group is the Z (i.e., syn)-configuration. Although the Z (i.e.,syn)-isomers of the invention are preferred and show the best biologicalproperties, they may coexist with small amounts of the E (oranti)-isomer which may exist due to isomerization during the chemicalpreparation.

According to the present invention, the cephalosporin compound (I) isprepared by the steps of (i) condensing a compound of the formula:##STR9## wherein R⁴ is a protecting group and R¹ and n are the same asdefined above, or a reactive derivative thereof with a compound of theformula: ##STR10## wherein R⁵ is carboxy or a protected carboxy when R²is acetoxy or (1-methyl-1H-tetrazol-5-yl)thio; or R⁵ is --COO⁻ when R²is a group of the formula: ##STR11## (Y is the same as defined above),or a salt thereof to give a compound of the formula: ##STR12## whereinR¹, R², R⁴, R⁵ and n are the same as defined above, and

(ii) removing the protecting group or groups therefrom.

Alternatively, the cephalosporin compound of the formula: ##STR13##wherein R⁶ is (1-methyl-1H-tetrazol-5-yl)thio or a group of the formula:##STR14## and R¹, R³, Y and n are the same as defined above, is preparedby the steps of

(i) reacting a compound of the formula: ##STR15## wherein R⁷ is hydrogenor a protecting group and R¹ and n are the same as defined above, or asalt thereof with a pyridine compound of the formula: ##STR16## (Y isthe same as defined above), (1-methyl-5-mercapto)tetrazol or a saltthereof, and

(ii) when R⁷ is a protecting group, further removing said protectinggroup therefrom.

In the above-mentioned reactions, a wide variety of protecting groupswhich have been usually employed to protect a amino group in the peptidesynthesis can be used as the protecting group R⁴ or R⁷. Examples of suchprotecting groups include lower alkanoyl such as formyl, acetyl andpivaloyl; mono-, di- or trihalogeno-lower alkanoyl such as chloroacetyland trifluoroacetyl; lower alkoxycarbonyl such as methoxycarbonyl,ethycarbonyl and tert.-butoxycarbonyl; substituted or unsubstitutedbenzyloxycarbonyl such as benzyloxycarbonyl andp-methoxybenzyloxycarbonyl; substituted or unsubstituted phenyl-loweralkyl such as p-methoxybenzyl and 3,4-dimethoxy-benzyl; and di- ortriphenyl lower alkyl such as benzhydryl and trityl. On the other hand,when R⁵ in the compound (III) or (IV) is a protected carboxy, theprotecting group on the carboxy group should be one which can be easilyremoved by conventional manner such as hydrolysis, acid treatment orreduction. Examples of such protecting groups include lower alkyl suchas methyl, ethyl or tert.-butyl; substituted or unsubstitutedphenyl-lower alkyl such as benzyl, p-methoxybenzyl and p-nitrobenzyl;benzhydryl; tri-lower alkylsilyl such as trimethylsilyl; and the like.When R⁵ is carboxy, it is preferred that the compound (III) is convertedto a salt thereof prior to carrying out the condensation reaction.Suitable examples of the salt of each one of the compound (III), thecompound (V) and (1-methyl-5-mercapto)tetrazole are inorganic salts suchas sodium or, potassium salts or organic salts such as trimethylamineor, triethylamine salts. Moreover, while the compound (II) can exist inthe form of two optical isomers due to the asymmetric carbon atominvolved in the group of the formula: ##STR17## (wherein the asteriskdenotes an asymmetric carbon atom), either an optical isomer of thecompound (II) or a racemic modification thereof may be used for thepurpose of the present invention. Throughout the specification andclaims, "levorotatory isomer" of the compound (I), (II), (IV) or (V) inwhich R¹ is hydrogen and n is an integer of 2 means that the absoluteconfiguration of said compound at said asymmetric carbon atoms is theS-configuration and also "dextrorotatory isomer" means that the absoluteconfiguration of said compound at said asymmetric carbon atom is theR-configuration.

The condensation reaction of the compound (II) or a reactive derivativethereof with the compound (III) or a salt thereof can be accomplished inconventional manner. For example, the condensation of the compound (II)in its free form with the compound (III) is conducted in the presence ofa dehydrating agent in a solvent. Suitable examples of the dehydratingagent include dicyclohexylcarbodiimide,N-cyclohexyl-N'-morpholinocarbodiimide,N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide, phosphorus oxychloride,phosphorus trichloride, thionyl chloride, oxalyl chloride,triphenylphosphine and the like. Vilsmeier reagent prepared fromdimethylformamide and phosphorus oxychloride, from dimethylformamide andoxalyl chloride, from dimethylformamide and phosgene or fromdimethylformamide and thionyl chloride may also be used as saiddehydrating agent. It is preferred to carry out the reaction at atemperature of -51° to 50° C., especially -30° to 20° C. Dioxane,tetrahydydrofuran, acetonitrile, chloroform, methylene chloride,dimethylformamide, N,N-dimethylacetamide, ethyl acetate, pyridine,acetone and water are suitable as the solvent.

On the other hand, the condensation reaction of the reactive derivativeof the compound (II) with the compound (III) or a salt thereof can beconducted either in the presence or absence of an acid acceptor in asolvent. Suitable examples of the reactive derivative of the compound(II) include the corresponding acid halides (e.g., chloride, bromide),mixed anhydrides (e.g., a mixed anhydride of the compound (II) withalkyl carbonate), active esters (e.g., p-nitrophenyl ester,2,4-dinitrophenyl ester, succinimide ester, phthalimide ester,benzotriazole ester, 2-pyrrolidon-1-yl ester), acid azide and acidamides (e.g., imidazole amide, 4-substituted-imidazole amide, triazoleamide). Dioxane, tetrahydrofuran, acetonitrile, chloroform, methylenechloride, dimethylformamide, N,N-dimethylacetamide, ethyl acetate,pyridine, acetone and water are suitable as the solvent. Moreover,suitable examples of the acid acceptor include alkali metal hydroxides(e.g., potassium hydroxide, sodium hydroxide), alkali metal carbonatesor bicarbonates (e.g., sodium carbonate, sodium bicarbonate, potassiumcarbonate, potassium bicarbonate), trialkyl amines (e.g.,trimethylamine, triethylamine), N,N-dialkylanilines (e.g.,N,N-dimethylaniline, N,N-diethylaniline), pyridine andN-alkyl-morphorlines (e.g., N-methylmorpholine). It is preferred tocarry out the reaction at a temperature of -50° to 50° C., especially at-30° to 20° C.

The removal of the protecting group or groups from the compound (IV)thus obtained can be conducted by a conventional manner such as, forexample, hydrolysis, solvolysis, acid treatment or reduction. Forexample, when the protecting group R⁴ is formyl, acetyl,tert.-butoxycarbonyl, benzhydryl or trityl and the protecting group onthe carboxy group is tert.-butyl or benzhydryl, said group or groups maybe removed by treating the compound (IV) with an acid. Suitable examplesof such acid include, for example, formic acid, trifluoroacetic acid,benzenesulfonic acid, p-toluene-sulfonic acid, hydrochloric acid orhydrogen bromide, especially trifluoroacetic acid. This reaction may beconducted with or without a solvent. Examples of the solvent are water,methanol, ethanol, acetic acid or dioxane. It is preferred to carry outthe reaction at a temperature of -30° to 70° C., especially 0° to 30° C.Moreover, when the trifluoroacetic acid is used as the acid, it ispreferred to carry it out in the presence of anisole. When theprotecting group R⁴ is benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,benzyl, p-methoxy-benzyl or 3,4-dimethoxybenzyl and the protecting groupon the carboxy group is benzyl, p-methoxybenzyl or p-nitrobenzyl, theremoval of said protecting group or groups may be conducted by catalytichydrogenation of the compound (IV) in hydrogen gas in the presence of acatalyst. This catalytic hydrogenation is preferably carried out at atemperature of 0° to 100° C., especially 10° to 40° C., underatmospheric or increased pressure. Preferred examples of the catalystinclude palladium-BaCO₃, palladium-charcoal and palladium-black.Methanol, ethanol, tetrahydrofuran and water are suitable as thereaction solvent. Further, when the protecting group R⁴ istrifluoroacetyl, pivaloyl, methoxycarbonyl or ethoxy-carbonyl and theprotecting group on the carboxy group is methyl or ethyl, said group orgroups may be removed by hydrolysis of the compound (IV). The hydrolysisof the compound (IV) can be carried out in a conventional manner, forexample, by treating it with an alkali agent such as sodium hydroxide orpotassium hydroxide, or an acid such as hydrochloric acid or hydrobromicacid. It is preferred to carry out said hydrolysis at a temperature of0° to 70° C., especially 10° to 30° C. When the protecting group R⁴ ischloroacetyl, said group may be removed by treating the compound (IV)with thiourea in a solvent. Methanol, ethanol and water are suitable asthe solvent. It is preferred to carry it out at a temperature of 20° to80° C., especially 40° to 80° C.

On the other hand, the reaction of the compound (V) or a salt thereofwith the pyridine compound ##STR18## or (1-methyl-5-mercapto)-tetrazoleor a salt thereof can be readily conducted in a solvent. Water,dimethylformamide and N,N-dimethylacetamide are suitable as the solvent.It is preferred to carry out the reaction at a temperature of 0° to 100°C., especially 20° to 80° C. It is also preferred to carry it out in thepresence of sodium iodide, potassium iodide, sodium bicarbonate orphosphate buffer solution. Removal of the protecting group R⁷ of theproduct thus obtained can be conducted in the same manner as in removingthe protecting group or groups from the compound (IV).

Concomitantly, the starting compound (II) of the present invention isprepared, for example, by reacting a compound of the formula: ##STR19##wherein R⁴ is the same as defined above, with a compound of the formula:##STR20## (wherein X is halogen and R¹ and n are the same as definedabove) in the presence of an alkali agent (e.g., potassium carbonate) ina solvent (e.g., dimethylsulfoxide) at a temperature of 10° to 50° C. togive a compound of the formula: ##STR21## wherein R¹, R⁴ and n are thesame as defined above, and then hydrolyzing the compound (VII).Alternatively, the starting compound (II) may be prepared by hydrolyzingthe compound (VI) to give a compound of the formula: ##STR22## whereinR⁴ is the same as defined above, and then reacting the compound (VIII)with a compound of the formula: ##STR23## (wherein R¹, n and X are thesame as defined above) in the presence of an acid acceptor (e.g., sodiumhydride) at a temperature of 10° to 40° C. in a solvent (e.g.,dimethylsulfoxide). Moreover, as mentioned hereinbefore, the compound(II) involves two optical isomers due to the asymmetric carbon involvedin the group of the formula: ##STR24## (wherein the asterisk denotes anasymmetric carbon atom). If required, however, such optical isomers maybe separated into each of the optical isomers by optical resolutionthereof. For example, the compound (II) in which R¹ is hydrogen, n is aninteger of 2 and R⁴ is trityl can be readily separated into each of theoptical isomers by reacting the racemic modification of the compound(II) with L- or D-phenylalanine methyl ester in a solvent (e.g., amixture of methanol and dioxane) to form the diastereoisomeric saltsthereof, and separating said diastereoisomers into each componentthereof by selective recrystallization. By said selectiverecrystallization, the least soluble diastereoisomer is recovered ascrystals from the reaction mixture and the more soluble diastereoisomerremains soluble therein. It is preferred to carry out said selectivecrystallization at a temperature of 10° to 40° C.

The cephalosporin compound (I) of the present invention andpharmaceutically acceptable salts thereof show potent antimicrobialactivity against a wide variety of microorganisms including thosebelonging to the genera Streptococcus (e.g., St. faecalis, St.pneumoniae), Staphylococcus (e.g., S. aureus, S. epidermidis) andPseudomonas (e.g., Ps. aeruginosa, Ps. putida, Ps. stutzeri), and areparticularly characterized by their potent antimicrobial activityagainst both gram-positive and gram-negative bacteria. For example,7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) and7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyiminoacetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylicacid exhibit minimum inhibitory concentration (M.I.C.) (Agar dilutionmethod, cultured for 20 hours at 37° C.) of 12.5 and 25 μg/ml againstStreptococcus faecalis CN 478, while the M.I.C. of Cefmenoxime [Chemicalname:7β-[(Z)-2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylicacid] and Ceftazidime [Chemical name:7β-[(Z)-2-(2-aminothiazol-4-yl)-2-(2-carboxyprop-2-yloxyimino)acetamido]-3-(1-pyridinomethyl)-3-cephem-4-carboxylate]against said microorganism are more than 100 μg/ml. The antimicrobialactivity of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thio-methyl]-3-cephem4-carboxylicacid against Staphylococcus aureus 252R is also more than 16 timesstronger than those of Cefmenoxime and Ceftazidime. Moreover, thecompound (I) and a salt thereof show potent antimicrobial activityagainst bacteria belonging to the genera Bacillus (e.g., B. subtilis),Escherichia (e.g., E. coil), Klebsiella (e.g., K. pneumoniae),Enterobacter (e.g., E. aerogenes, E. cloacae) and Serratia (e.g., S.marcescens).

The cephalosporin compound (I) may further exhibit potent antimicrobialactivity against other bacteria belonging to the genera Citrobacter,Proteus, Shigella, Hemophilus and Salmonella. Further, the cephalosporincompound (I) and salts thereof are characterized in that they showpotent protective effects against microbial infections of variousbacteria including both Staphylococcus aureus and Pseudomonasaeruginosa, because of the high absorbability or long-lastingtherapeutic effects in living tissues; that they have a high stabilityagainst a variety of β-lactamase-producing microorganisms, especiallyagainst β-lactamases produced by Proteus vulgaris; and also that theyare low in toxicity. For example, no rats die even after7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridinomethyl)-3-cephem-4-carboxylate(l-isomer) is administered subcutaneously to SD-male rats at a dose of1000 mg for 14 consecutive days.

The cephalosporin compound (I) of the present invention can be used forpharmaceutical use either in the free form or in the form of a saltthereof. Pharmaceutically acceptable salts of the compound (I) include,for example, non-toxic metallic salts such as sodium, potassium, calciumor aluminum salts; ammonium salt; salts thereof with non-toxic aminessuch as trialkylamines (e.g., triethylamine and procaine); salts thereofwith inorganic acids such as hydrochloric acid or hydrobromic acid;salts thereof with organic acids such as oxalic acid or tartaric acid;and so forth. These salts are easily obtained by treating the compound(I) with a stoichiometrically equi-molar amount of the correspondingalkali agent or acid at around room temperature in an aqueous solvent.The cephalosporin compound (I) and a salt thereof can be administeredeither orally or parenterally (e.g., intravenously, intramuscularly,subcutaneously). The daily dose of the compound (I) or a salt thereofmay vary over a wide range depending on the age, weight or conditions ofpatients, and the severity of diseases to be treated. In general,however, a preferred daily dose of said compound (I) or a salt thereofmay be about 0.002 to about 0.2 g, especially 0.01 to 0.04 g, per kg ofbody weight per day. Further, the compound (I) and a salt thereof may beused in the form of a pharmaceutical preparation containing the samecompound in conjunction or admixture with pharmaceutical excipientssuitable for oral or parenteral administration. Suitable excipientsinclude, for example, gelatin, lactose, glucose, sodium chloride,starch, magnesium stearate, talcum, vegetable oil and other knownmedicinal excipients. The pharmaceutical preparations may be in solidform such as tablets, coated tablets, pills or capsules; or in liquidform such as solutions, suspensions or emulsions. They may be sterilizedand/or may further contain auxiliaries such as stabilizing, wetting oremulsifying agents.

Practical and presently-preferred embodiments of the present inventionare illustratively shown in the following Examples. Throughout thespecification and claims, the term "lower alkyl" should be interpretedas referring to alkyl having one to four carbon atoms.

EXPERIMENT I (Antimicrobial activity in vitro)

The minimum inhibitory concentration (MIC, μg/ml of a test compound wasdetermined by means of a standard agar plate dilution method (based onthe standard method of Japan Society of Chemotherapy). Media used inthese experiments were Mueller-Hinton agar (MHA; Nissui).

The results are shown in the following Table 1.

                  TABLE 1                                                         ______________________________________                                                M.I.C. (μg/ml)                                                               The compound of the                                                                          Cef-                                                 Microorganisms                                                                          present invention                                                                            menoxime  Ceftazidime                                tested    (Note: 1)      (Note: 2) (Note: 3)                                  ______________________________________                                        Staphylococcus                                                                          0.78           1.56      12.5                                       aureus Terajima                                                               Staphylococcus                                                                          25             >100      >100                                       aureus 252R                                                                   Streptococcus                                                                           12.5           >100      >100                                       faecalis CN-478                                                               Bacillus subtilis                                                                       0.2            1.56      3.13                                       ATCC 6633                                                                     Klebsiella                                                                              0.05           0.1       0.1                                        pneumoniae 5038                                                               Enterobacter                                                                            ≦0.05   0.1       0.2                                        cloacae TU-680                                                                Serratia  ≦0.05   0.2       0.2                                        marcescens 7006                                                               Pseudomonas                                                                             0.39           6.25      0.78                                       aeruginosa 4096                                                               Pseudomonas                                                                             1.56           100       12.5                                       putida                                                                        ATCC 12633                                                                    ______________________________________                                         (Note: 1):                                                                    7{(Z)--2(2-aminothiazol-4-yl)-2-[(2pyrrolidon-3-yl)-oxyimino]acetamido3-(    -pyridiniomethyl)-3-cephem-4-carboxylate (lisomer)                             (Note: 2): Chemical name =                                                    7[(Z)--2(2-aminothiazol-4-yl)-2-methoxyimino)acetamido3-[(1methyl-1H--tet    azol5-yl)-thiomethyl3-cephem-4-carboxylic acid                                 (Note: 3): Chemical name =                                                    7[(Z)--2(2-aminothiazol-4-yl)-2-(2-carboxy-prop-2-yloxyimino)acetamido3-(    -pyridiniomethyl)-3-cephem-4-carboxylate                                  

EXPERIMENT II (Protective effects on bacterial infections in mice)

Ten male mice weighing 20±1 g were used for each dose level. Mice werechallenged via the intraperitoneal route with sufficient bacteria tokill all non-treated mice within 24 hours. All bacteria were suspendedin 6% mucin. A test compound were administered intramuscularly one hourafter the infection. Survival ratios were determined 7 days after theinfection. The median effective doses (ED₅₀, mg/kg) of the test compoundwere estimated by the probito analysis.

The results are shown in the following Table 2 together with the M. I.C. (μg/ml) of the test compound which was estimated in the same manneras described in Experiment I.

                  TABLE 2*                                                        ______________________________________                                                ED.sub.50 (mg/kg)                                                               The compound of the                                                                          Cef-                                                 Microorganisms                                                                          present invention                                                                            menoxime  Ceftazidime                                tested    (Note: 1)      (Note: 2) (Note: 3)                                  ______________________________________                                        Staphylococcus                                                                          1.71           5.32      7.85                                       aureus Smith                                                                            (1.56)         (1.56)    (12.5)                                     Escherichia                                                                             0.05           0.16      0.08                                       coli KC-14                                                                              (0.05)         (0.025)   (0.05)                                     Serratia  0.14           0.88      0.54                                       marcescens 7006                                                                         (0.05)         (0.2)     (0.2)                                      Citrobacter                                                                             0.06           0.19      0.15                                       freundii 916                                                                            (0.1)          (0.05)    (0.39)                                     Enterobacter                                                                            1.37           23.85     26.38                                      aerogenes 816                                                                           (0.39)         (1.56)    (6.25)                                     ______________________________________                                         Note:                                                                         *the numerical values in parenthesises show the M.I.C. (minimum inhibitor     concentration, μg/ml) of each test compound.                               (1)-(3): same as shown in the footnote of Table 1.                       

EXAMPLE 1

(1) 3.2 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid are suspended in 60 ml of tetrahydrofuran, and 2.05 g oftert.-butyl 7-aminocephalosporanate, 1.27 g of 1-hydroxybenzotriazoleand 1.93 g of dicyclohexylcarbodiimide are added thereto. The mixture isstirred at room temperature for 3 hours. Insoluble materials arefiltered off, and the filtrate is concentrated to dryness under reducedpressure. The residue is dissolved in ethyl acetate, and the solution iswashed with 1% hydrochloric acid, 5% aqueous sodium bicarbonate solutionand water, successively. The ethyl acetate solution is dried andconcentrated to dryness under reduced pressure. Then, the residue thusobtained is purified by silica gel chromatography (solvent, chloroform:methanol=98.5:1.5). 4.3 g of tert.-butyl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanateare obtained as pale yellow powder.

M.p. 135°-145° C. (decomp.).

NMR (CDCl₃)δ: 1.52 (9H, s), 2.02 (3H, s), 2.2-2.7 (2H, m), 3.0-3.5 (4H,m), 4.5-5.3 (4H, m), 5.6-6.0 (1H, m), 6.70 (1H, s), 6.9-7.5 (17H, m),8.4-8.7 (1H, broad).

(2) 1.0 g of tert.-butyl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanateis added to a mixture of 20 ml of trifluoroacetic acid and one ml ofanisole, and the mixture is stirred at room temperature for 20 minutes.The mixture is concentrated under reduced pressure to removetrifluoroacetic acid. Ether is added to the residue, and the resultingpowder is collected by filtration. The powder is suspended in 10 ml ofwater and sodium bicarbonate is added thereto to dissolve said powdertherein. The solution is washed with ethyl acetate and chromatographedon a column of non-ionic polymer resin Amberlite XAD-2 (registered trademark, manufactured by Rohm & Haas Co., U.S.A.), using water as aneluent. The fractions containing the cephalosporin compound as collectedand concentrated under reduced pressure to remove solvent. Then, acetoneis added to the residue obtained, and the resulting powder is collectedby filtration. 320 mg of sodium7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanateare obtained as colorless powder.

NMR (D₂ O)δ: 2.10 (3H, s), 2.2-2.7 (2H, m), 3.15-3.85 (4H, m), 4.6-5.00(2H, m), 5.01 (1H, t, J=7 Hz), 5.16 (1H, d, J=5 Hz), 5.77 (1H, d, J=5Hz), 6.98 (1H, s).

EXAMPLE 2

(1) 4.0 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid are dissolved in a mixture of 30 ml of tetrahydrofuran and 10 ml ofN,N-dimethylacetamide, and 1.27 g of 1-hydroxybenzotriazole and 1.93 gof dicyclohexylcarbodiimide are added thereto. After the mixture isstirred at 0° to 5° C. for 2 hours, said mixture is added to 30 ml ofN,N-dimethylacetamide-water (water content:15%) containing 2.12 g of7-aminocephalosporanic acid and 4 g of triethylamine. Said addition iscarried out under ice-cooling. Then, the mixture is stirred at the sametemperature for 1.5 hours. Insoluble materials are filtered off, and thefiltrate is concentrated under reduced pressure to remove solvent. Theresidue is poured into 300 ml of water. Said mixture is adjusted to pH 8with sodium bicarbonate, washed with ethyl acetate, adjusted to pH 3with 2N hydrochloric acid, and then extracted with ethyl acetate. Theextract is dried and concentrated to dryness under reduced pressure.Ether is added to the residue thus obtained, and the resulting powder iscollected by filtration. 3.1 g of7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid are obtained.

NMR (DMSO-d₆)δ: 2.03 (3H, s), 2.1-2.5 (2H, m), 3.0-3.7 (4H, m), 4.4-5.2(4H, m), 5.5-5.9 (1H, m), 6.71 (1H, s), 7.0-7.6 (15H, m), 7.84 (1H, s),8.80 (1H, broad s), 9.50 (1H, broad d).

(2) 40 ml of 80% aqueous formic acid are added to 3.0 g of7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-cephalosporanicacid, and the mixture is stirred at room temperature for 2 hours.Insoluble materials are filtered off, and the filtrate is concentratedto dryness under reduced pressure. Water is added to the residue, andthe aqueous mixture is neutralized with sodium bicarbonate and washedwith ether. Then, said aqueous mixture is chromatographed on a column ofnon-ionic polymer resin Diaion HP-20 (registered trade mark,manufactured by Mitsubishi Chemical Industries Ltd., Japan), using wateras an eluent. The fractions containing the cephalosporin compound arecollected and concentrated to dryness under reduced pressure. 1.5 g ofsodium7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-cephalosporanateare obtained.

The physico-chemical properties of this product are identical with thoseof the sample obtained in Example 1-(2).

EXAMPLE 3

(1) 3.25 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid are dissolved in 200 ml of tetrahydrofuran, and 3.14 g ofbenzhydryl7β-amino-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylate,1.03 g of 1-hydroxybenzotriazole and 1.57 g of dicyclohexylcarbodiimideare added thereto. The mixture is stirred at room temperature for 2hours. Insoluble materials are filtered off, and the filtrate isconcentrated to dryness under reduced pressure. The residue is dissolvedin ethyl acetate, and washed with 1% hydrochloric acid, 5% aqueoussodium bicarbonate solution and an aqueous saturated sodium chloridesolution, successively. The ethyl acetate solution is then dried andconcentrated to dryness under reduced pressure. The residue thusobtained is purified by silica gel chromatography (solvent, ethylacetate: benzene=5:2). 3.7 g of benzhydryl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylate are obtained.

M.p. 122°-126° C. (decomp.).

NMR (CDCl₃)δ: 2.2-2.7 (2H, m), 3.0-3.5 (2H, m), 3.6-3.75 (2H, m), 3.78(3H, s), 4.2-4.4 (2H, m), 5.03 (2H, m), 5.7-6.05 (1H, m), 6.75 (1H, s),6.88 (1H, s), 7.1-7.5 (27H, m), 8.80 (1H, broad).

(2) 860 mg of benzhydryl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-b5-yl)thiomethyl]-3-cephem-4-carboxylate are added to a mixture of 10 mlof trifluoroacetic acid and 0.5 ml of anisole, and the mixture isstirred at room temperature for 20 minutes. The mixture is concentratedunder reduced pressure to remove trifluoroacetic acid. Ether is added tothe residue, and the resulting powder is collected by filtration. Thepowder is suspended in water, and sodium bicarbonate is added thereto todissolve said powder therein. The solution is washed with ethyl acetateand chromatographed on a column of non-ionic polymer resin AmberliteXAD-2 (registered trade mark, manufactured by Rohm & Haas Co., U.S.A.),using water as an eluent. The fractions containing the cephalosporincompound are collected and concentrated to dryness under reducedpressure. 0.3 g of sodium7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylate is obtained.

NMR (DMSO-d₆)δ: 2.1-2.5 (2H, m), 3.1-3.5 (2H, m), 3.94 (3H, s), 4.2-4.5(2H, m), 4.6-4.8 (1H, m), 5.03 (1H, d, J=5 Hz), 5.5-5.8 (1H, m), 6.76(1H, s), 7.3 (2H, broad s), 8.00 (1H, s), 9.55 (1H, broad).

EXAMPLE 4

0.57 g of oxalyl chloride is added at -5° to 0° C. to 15 ml ofchloroform containing 0.35 g of dimethylformamide, and the mixture isstirred at the same temperature for 15 minutes. A mixture of 1.54 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid, 0.3 g of triethylamine and 15 ml of chloroform is added to saidmixture at -30° to -35° C., and the mixture is further stirred at thesame temperature for 5 minutes. Then, a solution of7β-amino-3-(1-pyridinio-methyl)-3-cephem-4-carboxylate in chloroform(said solution is prepared by suspending 1.82 g of the dihydrochlorideof said cephem compound in 10 ml of chloroform and adding 4 ml ofN,O-bis(trimethylsilyl)acetamide thereto to dissolve said salt therein)is added to said mixture at -35° to -30° C., and the mixture is stirredat the same temperature for 10 minutes and at -30° to -10° C. for onehour. The mixture is concentrated to dryness under reduced pressure. 60ml of 80% aqueous formic acid are added to the residue, and the aqueousmixture is stirred at room temperature for one hour. 50 ml of water areadded to said aqueous mixture. Then, insoluble materials are filteredoff, and the filtrate is washed with ethyl acetate and concentrated todryness under reduced pressure. The residue thus obtained is dissolvedin water and chromatographed on a column of non-ionic polymer resinDiaion HP-20 (registered trade mark, manufactured by Mitsubishi ChemicalIndustries Ltd., Japan). The column is washed with water, followed byelution with 20% methanol. The fractions containing the cephalosporincompound are collected and concentrated to dryness under reducedpressure. Acetone is added to the residue thus obtained, and theresulting powder is collected by filtration. 0.84 g of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylateis obtained.

M.p. >250° C.

NMR (D₂ O)δ: 2.2-2.7 (2H, m), 3.1-3.7 (4H, m), 4.9-5.5 (4H, m), 5.80(1H, d, J=5 Hz), 6.92 (1H, s), 7.8-9.1 (5H, m).

[α]_(D) ²⁰ +13.4° (C=1.0, H₂ O).

EXAMPLE 5

1.56 g of oxalyl chloride are added at -5° to 0° C. to 39 ml ofchloroform containing 0.99 ml of dimethylformamide, and the mixture isstirred at the same temperature for 15 minutes. A solution of 4.23 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (d-isomer) and 0.84 g of triethylamine in 39 ml of chloroform isadded to said mixture at -30° C. The mixture is stirred at the sametemperature for 5 minutes. Then, a solution of7β-amino-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate in chloroform(said solution is prepared by suspending 5.0 g of the dihydrochloride ofsaid cephem compound in 39 ml of chloroform and adding 11 ml ofN,O-bis(trimethylsilyl)acetamide thereto to dissolve said salt therein)is added to the said mixture at -30° to -10° C. After the mixture isstirred at the same temperature for 30 minutes, said mixture isconcentrated to dryness under reduced pressure. 100 ml of 80% aqueousformic acid are added to the residue, and said aqueous mixture isstirred at room temperature for one hour. 100 ml of water are added tothe mixture, and insoluble materials are filtered off. The filtrate iswashed with ethyl acetate and is concentrated to dryness under reducedpressure. The residue thus obtained is dissolved in water andchromatographed on a column of non-ionic polymer resin Diaion HP-20(resistered trade mark, manufactured by Mitsubishi Chemical IndustriesLtd., Japan). The column is washed with water, followed by elution with20% aqueous methanol. The fractions containing the cephalosporincompound are collected and concentrated to dryness under reducedpressure. Acetone is added to the residue, and the resulting powder iscollected by filtration. 2.14 g of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(d-isomer) are obtained. Another designation of this dextrorotatoryisomer is shown as7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[((3R)-2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate.

NMR (D₂ O)δ: 2.1-2.7 (2H, m), 3.1-3.7 (4H, m), 4.9-5.5 (4H, m), 5.79(1H, d, J=5 Hz), 6.92 (1H, s), 7.8-9.1 (5H, m).

[α]_(D) ²⁰ +45.7° (C=1, H₂ O).

EXAMPLE 6

1.81 g of oxalyl chloride are added at -5° to 0° C. to 45 ml ofchloroform containing 1.15 ml of dimethylformamide, and the mixture isstirred at the same temperature for 15 minutes. A solution of 4.90 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (l-isomer) and 0.97 g of triethylamine in 45 ml of chloroform isadded to said mixture at -30° C. The mixture is stirred at the sametemperature for 5 minutes. Then, a solution of7β-amino-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate in chloroform(said solution is prepared by suspending 5.8 g of the dihydrochloride ofsaid cephem compound in 45 ml of chloroform and adding 12.7 ml ofN,O-bis(trimethylsilyl)acetamide thereto to dissolve said salt therein)is added to the said mixture at -30° to -10° C. After the mixture isstirred at the same temperature for 30 minutes, said mixture isconcentrated to dryness under reduced pressure. 100 ml of 80% aqueousformic acid are added to the residue, and said aqueous mixture isstirred at room temperature for one hour. 110 ml of water are added tothe mixture, and insoluble materials are filtered off. The filtrate iswashed with ethyl acetate and is concentrated to dryness under reducedpressure. The residue thus obtained is dissolved in water andchromatographed on a column of non-ionic polymer resin Diaion HP-20(registered trade mark, manufactured by Mitsubishi Chemical IndustriesLtd., Japan). The column is washed with water, followed by elution with20% aqueous methanol. The fractions containing the cephalosporincompound are collected and concentrated to dryness under reducedpressure. Acetone is added to the residue, and the resulting powder iscollected by filtration. 2.22 g of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) are obtained. Another designation of this levorotatory isomeris shown as7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[((3S)-2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate.

NMR (D₂ O)δ: 2.2-2.7 (2H, m), 3.1-3.8 (4H, m), 5.05 (1H, t, J=7 Hz),5.28 (1H, d, J=5 Hz), 5.36 (1H, d, J=14 Hz), 5.63 (1H, d, J=14 Hz), 5.87(1H, d, J=5 Hz), 6,98 (1H, s), 8.10 (2H, t, J=7.5 Hz), 8.57 (1H, t,J=7.5 Hz), 8.98 (1H, d, J=7.5 Hz).

[α]_(D) ²⁰ -33.0° (C=1, H₂ O).

EXAMPLE 7

A mixture of 13 g of sodium iodide and 4 ml of water is stirred at 80°C. 3.6 g of pyridine and 3.2 g of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid are added to said mixture, and the mixture is stirred at 75° to 80°C. for one hour. After cooling, the reaction mixture is poured into 150ml of water and concentrated to dryness under reduced pressure. Theresidue is dissolved in 150 ml of water, and the solution is adjusted topH 1 with 2N hydrochloric acid. Insoluble materials are filtered off,and the filtrate is washed with ethyl acetate, adjusted to pH 6 with 2Nsodium hydroxide solution and concentrated to a total volume of 30 mlunder reduced pressure. The solution thus obtained is chromatographed ona column of non-ionic polymer resin Diaion HP-20 (registered trade mark,manufactured by Mitsubishi Chemical Industries Ltd., Japan). The columnis washed with water, followed by elution with 20% aqueous methanol. Thefractions containing the cephalosporin compound are collected andconcentrated to dryness under reduced pressure. Acetone is added to theresidue thus obtained, and the resulting powder is collected byfiltration. 0.67 g of 7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylateis obtained.

The physico-chemical properties of the product are identical with thoseof the sample obtained in Example 4.

EXAMPLE 8

(1) 1.5 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]aceticacid are dissolved in 50 ml of tetrahydrofuran, and 1.4 g of benzhydryl7β-amino-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylate,0.6 g of 1-hydroxybenzotriazole and 0.92 g of dicyclohexylcarbodiimideare added thereto. The mixture is stirred at room temperature for 2hours. Then, the reaction mixture is treated in the same manner asdescribed in Example 3-(1). 1.58 g of benzhydryl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateare obtained.

NMR (CDCl₃)δ: 2.2-2.6 (2H, m), 2.75 (3H, s), 3.1-3.5 (2H, m), 3.5-3.8(2H, m), 3.84 (3H, s), 4.2-4.4 (2H, m), 4.8-5.2 (2H, m), 5.8-6.1 (1H,m), 6.72 (1H, s), 6.90 (1H, s) 7.0-7.5 (25H, m), 8.5-8.8 (2H, broad).

(2) 1.5 g of benzhydryl7β-{(Z)-2-(2-trithylaminothiazol-4-yl9-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateare added to a mixture of 1.5 ml of anisole and 10 ml of trifluoroaceticacid, and the mixture is stirred at room temperature for 30 minutes. Themixture is concentrated under reduced pressure to remove trifluoroaceticacid. Ether is added to the residue, and the resulting powder iscollected by filtration. The powder is suspended in 15 ml of water, andsodium bicarbonate is added thereto to dissolve said powder therein. Thesolution is washed with ethyl acetate and chromatographed on a column ofnon-ionic polymer resin Amberlite XAD-2 (registered trade mark,manufactured by Rohm & Haas Co., U.S.A.), using water as an eluent. Thefractions containing the cephalosporin compound are collected andconcentrated to dryness under reduced pressure. 0.61 g of sodium7β-{(Z)-2-(2-aminothizol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateis obtained.

NMR (D₂ O)δ: 2.1-2.7 (2H, m), 2.89 (3H, s), 3.2-3.8 (4H, m), 4.05 (3H,s), 4.05-4.3 (2H, m), 4.9-5.3 (2H, m), 5.73 (1H, d, J=5 Hz), 7.00 (1H,s).

EXAMPLE 9

(1) 0.5 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-piperidon-3-yl)oxyimino]aceticacid is dissolved in 10 ml of tetrahydrofuran, and 0.47 g of benzhydryl7β-amino-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylate,0.15 g of 1-hydroxybenzotriazole and 0.24 g of dicyclohexylcarbodiimideare added thereto. The mixture is stirred at room temperature for 2.5hours. The reaction mixture is treated in the same manner as describedin Example 3-(1). 0.47 g of benzhydryl7β-[(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-piperidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateis obtained.

NMR (DMSO-d₆)δ: 2.00-2.5 (4H, m), 3.0-3.3 (2H, m), 3.6-3.8 (2H, m), 3.8(3H, s), 4.1-4.3 (2H, m), 4.6-4.8 (1H, m) 4.9-5.4 (1H, m), 5.85 (1H, d,d, J=8 Hz, J=5 Hz), 6.71 (1H, s), 6.8, (1H, s) 7.0-7.6 (26H, m), 9.63(1H, d, J=9 Hz).

(2) 0.4 g of benzhydryl7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-piperidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateis added to a mixture of 10 ml of trifluoroacetic acid and 0.8 ml ofanisole, and the mixture is allowed to stand at room temperature for 30minutes. The mixture is evaporated under reduced pressure to removetrifluoroacetic acid. Ether is added to the residue, and the resultingpowder is collected by filtration. The powder is suspended in water, andsodium bicarbonate is added thereto to dissolve said powder therein. Thesolution is washed with ethyl acetate and chromatographed on a column ofnon-ionic polymer resin Amberlite XAD-2 (registered trade mark,manufactured by Rohm & Haas Co., U.S.A.) The column is washed withwater, followed by elution with 10% aqueous methanol. The fractionscontaining the cephalosporin compound are collected and concentrated todryness under reduced pressure. 0.9 g of sodium7β-{(Z)-2-(2-aminothizaol-4-yl)-2-[(2-piperidon-3-yl)oxyimino]acetamido}-3-[(1-methyl-1H-tetrazol-5-yl)thiomethyl]-3-cephem-4-carboxylateis obtained.

NMR (D₂ O)δ: 1.5-2.3 (4H, m), 3.0-3.4 (2H, m), 3.4-3.6 (2H, m), 3.90 (H,m), 4.0-4.2 (2H, m), 5.03 (1H, d, J=5 Hz), 5.63 (1H, d, J=5 Hz), 5.85(1H, s).

EXAMPLE 10

(1) 18.2 g of phosphorus oxychloride are added dropwise to 9.2 ml ofdimethylformamide under ice-cooling, and the mixture is stirred at 25°to 35° C. for 30 minutes. After cooling to 0° C., 100 ml of chloroformare added to the mixture, and said mixture is further cooled to -35° C.A solution of 20 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)-oxyimino]aceticacid (l-isomer) and 5.6 ml of triethylamine in 160 ml ofN,N-dimethylacetamide is added dropwise to the mixture at -35° to -25°C., and the mixture is stirred at the same temperature for 20 minutes.Then, a solution of 7-aminocephalosporanic acid (said solution beingprepared by stirring a mixture of 16 g of 7-aminocephalosporanic acid,48 g of trimethyl chlorosilane, 35.6 ml of pyridine and 160 ml of N,Ndimethylacetamide at 10° to 20° C. for 2 hours) is added dropwise to thereaction mixture at -35° to -20° C. under stirring. After stirring atthe same temperature for 20 minutes, said reaction mixture is pouredinto 2 liters of ice-water and stirred vigorously. Crystallineprecipitates are collected by filtration, washed with water, ethylacetate and ether, and then dried in vacuo. 26.5 g of7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-cephalosporanicacid (l-isomer) are obtained as colorless powder.

NMR (DMSO-d₆)δ: 2.06 (3H, s), 2.1-2,5 (2H, m), 3.1-3.4 (2H, m), 3.45(1H, d, J=18 Hz), 3,75 (1H, d, J=18 Hz) 4.6-5.02 (3H, m), 5.17 (1H, d,J=5 Hz), 5.6-5.9, (1H, m), 6.81 (1H, s), 7.1-7.6 (15H, m), 7.97 (1H, s),8.88 (1H, br, s), 9.64 (1H, d, J=6 Hz).

(2) 67.5 g of sodium iodide and 10.9 g of pyridine are added to amixture of 18 ml of water and 18 ml of dimethylformamide, and themixture is heated to 80° C. 11.5 g of7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid (l-isomer) are added to the mixture. The mixture is stirred at 80°C. for 35 minutes. After cooling, 90 ml of ice-water are added to thereaction mixture, and said mixture is washed with ethyl acetate. Then,the reaction mixture is evaporated under reduced pressure to removeethyl acetate and adjusted to pH 2 with 10% hydrochloric acid.Crystalline precipitates are collected by filtration, washed with waterand dried in vacuo. 11.9 g of7β-{(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) are obtained as crude pale yellow powder.

(3) 5.5 g of7β-{(Z)-2-(2-tritylamino-thiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) obtained above are dissolved in 90 ml of 80% formic acid. Thesolution is stirred at room temperature for one hour. The reactionsolution is concentrated under reduced pressure, and 200 ml of water areadded to the residue. Insoluble materials are filtered off. The aqueousfiltrate is washed with ethyl acetate, concentrated under reducedpressure to remove ethyl acetate and then introduced into a columnpacked with 200 ml of a non-ionic adsorption resin (manufactured byMitsubishi Chemical Industries Ltd. under the trade name "DiaionHP-20"). After the column is washed with water, said column is elutedwith 20% aqueous methanol. The eluate is concentrated under reducedpressure, acetone is added to the residue and the resultant precipitatesare collected by filtration. 1.6 g of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) are obtained as pale yellow powder.

The physicochemical properties of this product are identical with thoseof the sample obtained in Example 6.

EXAMPLE 11

2.5 g of7β-{(Z)-2-(2-tritylamino-thiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino)]acetamido}cephalosporanicacid (l-isomer) obtained in the same manner as described in Example10-(1) are dissolved in 50 ml of 80% formic acid, and the solution isstirred at room temperature for one hour. Insoluble materials arefiltered off. The filtrate is concentrated under reduced pressure, wateris added to the residue, and the aqueous mixture is neutralized withsodium bicarbonate. Then, said aqueous mixture is washed with ethylacetate, concentrated under reduced pressure and introduced into acolumn packed with 200 ml of a non-ionic absorption resin ("DiaionHP-20"). After the column is washed with water, said column is treatedin the same manner as described in Example 10-(3). 1.0 g of sodium7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanate(l-isomer) is obtained as pale yellow powder. Another designation ofthis levorotatory isomer is shown as sodium7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[((3S)-2-pyrrolidon-3-yl)-oxyimino]acetamido}cephalosporanate.

NMR (D₂ O)δ: 2.12 (3H, s), 2.3-2.6 (2H, m), 3.2-3.9 (4H, m), 4.75 (1H,d, J=13 Hz), 4.95 (1H, d, J=13 Hz), 5.10 (1H, t, J=7 Hz), 5.25 (1H, d,J=5 Hz), 5.85 (1H, d, J=5 Hz), 7.08 (1H, s).

The product obtained above is treated in the same manner as described inExample 7, whereby7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}-3-(1-pyridiniomethyl)-3-cephem-4-carboxylate(l-isomer) is obtained.

EXAMPLE 12

A mixture of 960 mg of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid, 730 mg of isonicotinamide, 4.5 g of sodium iodide and 3 ml ofwater is stirred at 65° to 70° C. for 6 hours. After cooling, 20 ml ofwater are added to the reaction mixture, and insoluble materials arefiltered off. The filtrate is introduced into a column packed with 100ml of a non-ionic adsorption resin ("Diaion HP-20"). The column iswashed with water. Then, said column is eluted with 20% aqueousmethanol, and the eluate is concentrated under reduced pressure. 175 mgof7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)-oxyimino]acetamido}-3-(4-carbamoyl-1-pyridiniomethyl)-3-cephem-4-carboxylateare obtained as pale yellow powder.

M.p. 163°-166° C. (decomp.).

NMR (CF₃ CO₂ D)δ: 2.5-3.0 (2H, m), 3.5-4.0 (4H, m), 5.2-5.5 (3H, m),5.5-5.8 (1H, m), 5.9-6.2 (1H, m), 7.35 (1H, s), 8.5-8.7 (2H, m), 9.1-9.4(2H, m).

EXAMPLE 13

A mixture of 960 mg of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid, 650 mg of 4-hydroxymethylpyridine, 4.5 g of sodium iodide and 3 mlof water is stirred at 65° to 70° C. for 7 hours. Then, the reactionmixture is treated in the same manner as described in Example 12. 150 mgof7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]-acetamido}-3-(4-hydroxymethyl-1-pyridiniomethyl)-3-cephem-4-carboxylateare obtained as pale yellow powder.

m.p. 162°-184° C. (decomp.).

NMR (D₂ O)δ: 2.3-2.7 (2H, m), 3.1-3.7 (4H, m), 4.8-5.0 (2H, m), 5.03(2H, s), 5.24 (1H, d, J=5 Hz), 5.3-5.6 (1H, m), 5.83 (1H, d, J=5 Hz),6.81 (1H, s), 7.8-8.2 (2H, m), 8.6-9.0 (2H, m).

EXAMPLE 14

A mixture of 960 mg of7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetamido}cephalosporanicacid, 650 mg of 3-hydroxymethylpyridine, 4.5 g of sodium iodide and 3 mlof water is treated in the same manner as described in Example 12. 83 mgof7β-{(Z)-2-(2-aminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]-acetamido}-3-(3-hydroxymethyl-1-pyridiniomethyl)-3-cephem-4-carboxylateare obtained.

M.p. 128°-135° C. (decomp.).

NMR. (D₂ O)δ: 2.1-2.8 (2H, m), 3.1-3.8 (4H, m), 4.86 (2H, s), 4.9-5.1(2H, m), 5.27 (1H, d, J=5 Hz), 5.3-5.6 (1H, m), 5.83 (1H, d, J=5 Hz),6.96 (1H, s), 7.7-8.2 (1H, m), 8.3-8.6 (1H, m), 8.6-9.1 (2H, m).

Preparation of Starting compounds

(1) 15.8 g of ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-hydroxyiminoacetate are dissolved in70 ml of dimethylsulfoxide, and 5.8 g of anhydrous potassium carbonateare added thereto. The mixture is stirred at room temperature for 20minutes. 6.6 g of 3-bromo-2-pyrrolidone are added to said mixture, andthe mixture is stirred at room temperature for 20 hours. The mixture ispoured into 800 ml of water, and crystalline precipitates are collectedby filtration and washed with water. The crystals are dissolved inchloroform, washed with water and then dried. Then, the chloroformsolution is evaporated under reduced pressure to remove solvent. 100 mlof ethyl acetate was added to the residue, and allowed to stand at roomtemperature. Crystalline precipitates thus obtained are collected byfiltration and dried. 16.0 g of ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetateare obtained.

M.p. 209°-210° C.

NMR (CDCl₃)δ: 1.30 (3H, t, J=7 Hz), 2.1-2.6 (2H, m), 3.1-3.6 (2H, m),4.34 (2H, q, J=7 Hz), 4.90 (1H, t, J=7 Hz), 6.53 (1H, s), 7.0∝7.6 (17H,m).

16.0 g of ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]acetateare added to a mixture of 160 ml of methanol and 30 ml of an aqueous 2Nsodium hydroxide solution, and the mixture is refluxed for 30 minutesunder heating. After cooling, crystalline precipitates are collected byfiltration and washed with methanol. The crystals are suspended in 30 mlof water. Then, the suspension is adjusted to pH 3 with 2N hydrochloricacid. Crystalline precipitates are collected by filtration and dried.11.4 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid are obtained.

M.p. 150°-153° C. (decomp.)

NMR (DMSO-d₆)δ: 1.8-2.4 (2H, m), 2.9-3.4 (2H, m), 4.63 (1H, t, J=7 Hz),6.76 (1H, s), 6.9-7.6 (15H, m), 7.85 (1H, s), 8.70 (1H, broad s).

(2) 30 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid and 60 ml of methanol are added to 100 ml of dioxane containing10.5 g of methyl L-phenylalaninate, and the mixture is heated at 50° C.to dissolve said acid therein. 700 ml of dioxane are added to thesolution, and the mixture is stirred at room temperature for 5 hours.Crystalline precipitates are collected by filtration (the filtrate ishereinafter referred to as "Filtrate I"), and 14.3 g of the crudeproduct thus obtained are dissolved in 24 ml of methanol. 280 ml ofdioxane are added to the methanol solution. The mixture is stirred atroom temperature for 4 hours, and crystalline precipitates are collectedby filtration (the filtrate is hereinafter referred to as "FiltrateII"). 12.2 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (l-isomer) methyl L-phenylalaninate salt are obtained.

[α]_(D) ²⁵ -14.0° (C=1, methanol).

12.2 g of the above-mentioned salt are dissolved in 120 ml of methanol,and 176 ml of 0.1N hydrochloric acid are added thereto. The mixture isstirred for 2 hours under ice-cooling. Crystalline precipitates arecollected by filtration and washed with methanol. 7.5 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (l-isomer) are obtained. Another designation of this levorotatoryisomer is shown as(Z)-2-(2-tritylaminothiazol-4-yl)-2-[((3S)-2-pyrrolidon-3-yl)oxyimino]aceticacid.

M.p. 142°-143° C. (decomp.).

[α]_(D) ²⁵ -38.8° (C=1, dimethylformamide).

(3) Filtrates I and II obtained in the above mentioned paragraph (2) arecondensed to dryness under reduced pressure. The residue is dissolved in250 ml of methanol and then 450 ml of 0.1N hydrochloric acid are addeddropwise to the solution. The mixture is stirred for 2 hours underice-cooling. The resulting crystalline precipitates are collected byfiltration, washed with methanol, and dried. 20 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (containing excess of the d-isomer) are obtained. 20.0 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid thus recovered and 40 ml of methanol are added to 70 ml of dioxanecontaining 7.0 g of methyl D-phenylalaninate, and the mixture is heatedat 50° C. to dissolve said acid therein. 450 ml of dioxane are added tosaid solution. Then, the mixture is stirred at room temperature for 4hours, and crystalline precipitates are collected by filtration. 13.3 gof the crude product thus obtained are dissolved in 20 ml of methanol,and 260 ml of dioxane are added thereto. The mixture is stirred at roomtemperature for 4 hours. Crystalline precipitates are collected byfiltration. 12.0 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (d-isomer) methyl D-phenylalaninate salt are obtained.

[α]_(D) ²⁵ +13.9° (C=1, methanol).

12.0 g of the above-mentioned salt are dissolved in 120 ml of methanol,and 174 ml of 0.1N hydrochloric acid are added thereto. The mixture isstirred for 2 hours under ice-cooling. Crystalline precipitates arecollected by filtration and washed with methanol. 7.3 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-pyrrolidon-3-yl)oxyimino]aceticacid (d-isomer) are obtained. Another designation of this dextrorotatoryisomer is shown as(Z)-2-(2-tritylaminothiazol-4-yl)-2-[((3R)-2-pyrrolidon-3-yl)oxyimino]aceticacid.

M.p. 143°-144° C. (decomp.).

[α]_(D) ²⁵ +37.4° (C=1, dimethylformamide).

(4) 2.7 g of ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-hydroxyiminoacetate are dissolved in12 ml of dimethylsulfoxide, and 1.0 g of anhydrous potassium carbonateis added thereto under nitrogen gas atmosphere. The mixture is stirredat room temperature for 10 minutes. 1.2 g of1-methyl-3-bromo-2-pyrrolidone are added to the mixture, and the mixtureis stirred at room temperature for 5 hours. The reaction mixture ispoured into 100 ml of water, and crystalline precipitates are collectedby filtration. The crystals are dissolved in ethyl acetate, and thesolution is washed with water and then dried. The solution isconcentrated under reduced pressure to remove solvent. Then, the residueis crystallized with isopropyl ether and collected by filtration 2.1 gof ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]acetateare obtained.

NMR (CDCl₃)δ: 1.30 (3H, t, J=7 Hz), 2.0-2.7 (2H, m), 2.88 (3H, s),3.0-3.6 (2H, m), 4.34 (2H, q, J=7 Hz), 4.92 (1H, t, J=7 Hz), 6.54 (1H,s), 6.87 (1H, s), 7.0-7.5 (15H, m).

2.7 g of ethyl(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino)acetateare suspended in 27 ml of methanol, and 4.9 ml of 2N sodium hydroxidesolution are added thereto. The mixture is refluxed for 20 minutes underheating. After cooling, the mixture is concentrated under reducedpressure to remove methanol. The residue is adjusted to pH 3 with 2Nhydrochloric acid and extracted with ethyl acetate. The extract is driedand evaporated under reduced pressure to remove solvent. Then, theresidue thus obtained is crystallized with ether and collected byfiltration. 2.15 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(1-methyl-2-pyrrolidon-3-yl)oxyimino]aceticacid are obtained.

M.p. 142°-145° C. (decomp.).

NMR (DMSO-d₆)δ: 2.0-2.5 (2H, m), 2.77 (3H, s), 3.1-3.4 (2H, m), 4.78(1H, t, J=8 Hz), 6.87 (1H, s), 6.9-7.5 (16H, m).

(5) 1.3 g of (Z)-2-(2-tritylaminothiazol-4-yl)-2-hydroxyliminoaceticacid are dissolved in 10 ml of dimethylformamide, and 0.24 g of sodiumhydride (60% oil dispersion) is added thereto. The mixture is stirred atroom temperature for 15 minutes. 0.65 g of 3-bromo-2-piperidone is addedto the mixture, and the mixture is stirred at room temperature for 1.5hours. The reaction mixture is poured into water and washed with amixture of ethyl acetate and tetrahydrofuran (1:1). The aqueous layer isadjusted to pH 3 with 10% hydrochloric acid and extracted with a mixtureof ethyl acetate and tetrahydrofuran (1:1). The extract is dried andconcentrated to dryness under reduced pressure. Then, ether is added tothe residue, and the resulting powder is collected by filtration. Thepowder (1.3 g) is purified by silica gel chromatography (solvent,methanol:chloroform=1:4). 0.85 g of(Z)-2-(2-tritylaminothiazol-4-yl)-2-[(2-piperidon-3-yl)oxyimino]aceticacid are obtained.

M.p. 145°-150° C. (decomp.).

What we claim is:
 1. A compound of the formula: ##STR25## wherein R¹ ishydrogen or a lower alkyl group;R⁴ is a protecting group; n is aninteger of 2 or 3 and X is an acid or base hydrolyzable group.
 2. Thecompound of claim 1, wherein X is --C₂ H₅.
 3. The compound of claim 1,wherein X is a hydrolyzable group which can be removed by treatment withan alkali agent.
 4. The compound of claim 1, wherein R⁴ is a protectinggroup which can be removed by hydrolysis, solvolysis, acid treatment orreduction.
 5. The compound of claim 1, wherein R⁴ is formyl, loweralkanoyl, mono-, di- or trihalogeno-lower alkanoyl, loweralkoxycarbonyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,phenyl-lower alkyl, p-methoxyphenyl-lower alkyl, dimethoxyphenyl-loweralkyl, or di or triphenyl-lower alkyl.